Use of long-wavelength electromagnetic radiation and photoprotective tumor localizing agents for diagnosis

ABSTRACT

A process of pathology or target tissue identification comprising administering an imaging material to a pathology or target tissue bearing mammalian host and irradiating the mammalian host with electromagnetic radiation having a wavelength between about 600 and 1100 nm, and especially in the region of 700 nm and longer wavelengths, whereupon the imaging material, which has been preferentially taken up by the pathology or target tissue, emits light and permits precise identification of the location, size and/or shape of the pathology or target tissue.

This is a divisional of application Ser. No. 09/282,610 filed Apr. 1,1999, now U.S. Pat. No. 6,183,727 which is a continuation of applicationSer. No. 09/081,175 filed May 19, 1998 now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a method of diagnosing mammalian pathology ortarget tissues and more particularly to a photoidentification method ofdiagnosing pathology or target tissue using an optical imaging materialcontaining an imaging agent and at least one auxiliary chromophore. Theimaging material preferentially localizes in pathology or target tissue,absorbs light and in some cases fluoresces or phosphoresces uponexposure to light. The primary purpose of the auxiliary chromophore isprevention of photodamage to healthy tissue by the agent.

Certain classes of molecules, including, for example, syntheticporphyrin derivatives, naturally occurring porphyrins and theirderivatives, chlorophylls and their derivatives, purpurins,phthalocyanines, other cyclic tetrapyrroles, and fullerenes can act asimaging, detection and diagnostic agents for pathologies or targettissues including tumors, atherosclerotic and arthritic tissue, anddiseased blood vessels. Administration of these agents to a human orother organism results in preferential localization of the agent in anyof a variety of pathologies with respect to surrounding tissue.Irradiation of the organism with light of a given wavelength orwavelengths results in absorption of light by the agent. In some cases,the agent then emits light by fluorescence or phosphorescence. Lightabsorption or light emission produces contrast between the pathology ortarget tissue and the surrounding tissue, and the detection of thiscontrast allows pathology or target tissue imaging, detection ordiagnosis. Alternatively, agents of this type can be used to enhancecontrast or otherwise improve detection in magnetic resonance imaging ofpathology or target tissues or can bear radioactive isotopes whosedetection can be the basis of pathology or target tissue imaging,detection and diagnosis, or can serve as contrast agents for X-rayradiological or other techniques involving high-energy radiation.

Absorption of light by the agent results in production of excitedstates. These excited states are by definition of higher energy than theoriginal unexcited ground state of the agent. The excess energy canresult in deleterious interactions with the organism. For example,excited triplet states of the agent (or singlet or other excited states)can react directly with tissue or other components of the organism tocause damage to the organism. Triplet states and other states of highmultiplicity can also cause the formation of excited states of oxygen,such as singlet oxygen, and other powerful oxidizing agents,superoxides, and other oxygen radicals. These excited states of oxygenand oxygen radicals are known to cause damage to biological membranes aswell as other components of the organism. The agent in an excited statecan also react with other molecules present to create other species thatare harmful to the organism. Such damage is not limited to pathology ortarget tissue, as the agent does not localize exclusively in thepathology or target tissue. After administration some agent is foundthroughout the organism, including the skin. This propensity to causedamage to healthy tissue in the organism can limit the usefulness of theagent for pathology or target tissue imaging detection and diagnosis.

Carotenoid pigments, which are ubiquitous in photosynthetic membranes,are essential for the survival of green plants. Three facets ofcarotenoid function are recognized in photosynthetic membranes. First,carotenoids photoprotect by rapidly quenching chlorophyll triplet stateswhich are formed in antenna systems or photosynthetic reaction centers.This triplet-triplet energy transfer preventschlorophyll-photosensitized formation of highly destructive singletoxygen which is injurious to the organism. In addition, carotenoids actas antennas by absorbing light in spectral regions where chlorophyllabsorbs weakly and by delivering the resulting excitation to chlorophyllvia a singlet-singlet energy transfer process. Finally, nearbycarotenoids quench chlorophyll first excited singlet states. Thisquenching has been ascribed to energy transfer or electron transfer orsome other process leading to internal conversion and is believed toplay a role in the regulation of photosynthesis.

A number of porphyrin materials have been found to localize inpathologies and damage that tissue upon irradiation with light. Many ofthese, such as “hematoporphyrin derivative” and related materials, arebeing investigated as photodynamic therapeutic agents. All of theseagents suffer from the problem that they are also absorbed by healthytissue, which is consequently harmed by light.

Various synthetic carotenoids designed to mimic carotenoid photoprotection have been investigated. Synthetic carotenoporphyrinsconsisting of a carotenoid part covalently linked to a syntheticmeso-tetraarylporphyrin which successfully exhibited the photophysicalfunctions of cartenoids in photosynthesis were first reported by G.Dirks, A. Moore, T. Moore and D. Gust in Photochemistry andPhotobiology, Vol. 32, pp. 277-280 (Permagon Press Ltd. Great Britain,1980).

A carotenoporphyrin which demonstrated quenching of the porphyrintriplet state by the attached carotenoid via triplet-triplet energytransfer was reported by R. V. Bensasson, E. J. Land, A. L. Moore, R. L.Crouch, G. Dirks, T. A. Moore and D. Gust in Nature, Vol. 290, No. 5804,pp. 329-332 (Mar. 16, 1981). Since that time, various compounds whichexhibit such triplet-triplet energy transfer have been reported. In1984, five carotenoporphyrins were prepared by Dr. Paul Liddell atArizona State University and reported in his doctoral thesis datedDecember 1985. Three carotenoporphyrins were reported by H. Frank, B.Chadwick, J. Oh, and D. Gust et al. in Biochemical et Biophysical Acta892 (1987), pp. 253-263.

It has also been previously shown in U.S. Pat. No. 5,286,474,incorporated by reference herein, that certain syntheticcarotenoporphyrins preferentially localize in mammalian pathology ortarget tissue where they absorb and emit light when irradiated withlight so that the site of the pathology or target tissue may be detectedby the fluorescence of the localized carotenoporphyrin.

SUMMARY OF THE INVENTION

This invention comprises the use of at least one auxiliary chromophore,such as carotenoids and other polyenes, to prevent undesired damage totissue that can be caused by agents, as described above. The auxiliarychromophore is placed in the vicinity of the agent, through chemicalbonding or other means, in such a way that it rapidly removes theexcitation energy of the agent before that energy can cause substantialdamage to the organism or sensitize the formation of singlet oxygen orother harmful species. The energy is removed through triplet—tripletenergy transfer, single-singlet energy transfer, or other quenchingphenomena The energy thus acquired by the auxiliary chromophore is in aform that is essentially harmless to the organism, and is rapidlydissipated in a harmless way.

Localization of the imaging material employed in the practice of thisinvention is advantageous over the use of porphyrins alone. In addition,photodamage of tissue is advantageously precluded by the quenching ofthe porphyrin triplet state, and most importantly, photopenetration ofthe tissue by excitation light and by emitted light is enhanced by theuse of agents that absorb and emit lower energy (longer wavelength)electromagnetic radiation. Thus this invention overcomes the problem ofcollateral tissue damage inherent with the use of existingphotosensitizing compounds as diagnostic agents, and increases thedetection of pathology or target tissues by using longer wavelengthelectromagnetic radiation.

This invention also provides a method of locating and visualizingmammalian pathology or target tissue. The method comprises administeringa diagnostically effective amount of an imaging material, comprising anagent and at least one auxiliary chromophore, to a mammalian host,permitting the diagnostic agent to localize in the pathology or targettissue and thereafter irradiating the mammalian host with low energyelectromagnetic radiation having a wavelength between about 600 and 1100nm. The localized imaging material thus absorbs and in some casesfluoresces, or otherwise luminesces sharply defining the pathology ortarget tissue. Light absorbed by or emitted from the pathology or targettissue by the localized diagnostic agent employed in this inventionsharply defines the location of the pathology or target tissue to beremoved or otherwise treated. At the same time, the auxiliarychromophore prevents or limits photodamage to healthy tissue byquenching excited triplet states of the agent. In the event that theimaging process involves a process other than absorption or emission oflight, the auxiliary chromophore still provides protection fromphotodamage by adventitious light in any tissues.

Generally, the imaging materials of the present invention may beeffectively administered to representative mammals in dosages of from0.5 to 50 μmol/kg of host body weight, preferably from 3 to 48 hoursprior to the diagnostic procedure or surgery.

The imaging materials employed in this invention have a number ofadvantages over current contrast agents and pathology or target tissuediagnostic procedures. They lack toxicity due to the antioxidantbehavior of the auxiliary chromophore and the use of lower energy,higher wavelength electromagnetic radiation. Many individuals aresensitive to present X-ray contrast agents employed, for example incomputer assisted tomography (CAT) scans, and there have been cases ofsevere allergy reactions resulting in anaphylactic shock. In addition,the patient is exposed to ionizing radiation. Alternatively, nuclearscans are often employed. However, nuclear scans require theadministration of radioactive diagnostic materials and further, areuseful primarily to define function as opposed to structure. Magneticresonance imaging is accurate and definitive for the diagnosis of brainand other abnormalities but is expensive, unpleasantly noisy, confiningfor claustrophobic individuals, and often times the contrast agentenhancement is not target specific. This invention is advantageous overthese alternative techniques. Lower cost is an additional advantage ofthis invention.

The present invention also provides an improved, more convenient andeconomical synthesis of the diagnostic agents employed herein.

DETAILED DESCRIPTION OF THE INVENTION

An imaging material comprising at least two parts is employed in thisinvention. One part is an agent which localizes in pathology or targettissue in preference to surrounding tissue, absorbs light to produce anexcited state, and allows imaging, demarcation, detection or diagnosisof the pathology or target tissue, or combinations thereof, via theabsorption of light, or the emission of fluorescence, phosphorescence orheat. These effects can be detected via absorption spectroscopy,fluorescence or phosphorescence spectroscopy, or heat detection bycalorimetric methods such as photothernal, photoacoustic, or mirageeffect. Alternatively, the agent can enhance imaging as a magneticresonance imaging contrast agent or by bearing radioactive isotopes ofany of various elements whose radioactive emissions can be detected andused for imaging and diagnostic purposes, or by acting as a contrastagent for imaging by X-radiation or other high-energy radiation. Thesecond part comprises an auxiliary chromophore, such as a carotenoid,polyene, or other chromophore with a low-energy triplet, singlet orcharge-separated state, which is held in the vicinity of the agentthrough chemical bonding or other mechanisms, and which prevents damageto the organism by quenching high-energy states of the agent viatriplet-triplet energy transfer, singlet-singlet energy transfer,electron transfer, or similar mechanisms.

Examples of preferred imaging materials include

wherein

R₁=hydrogen, alkyl or alkoxy groups; optionally the alkyl or alkoxygroups may include other groups such as COOH groups and the like,

R₂=alkyl or aryl groups; the alkyl or aryl groups may include othergroups such as COOH groups, NH₂ groups and the like;

wherein

R₁=hydrogen, alkyl or alkoxy groups; the alkyl or alkoxy groups mayinclude other groups such as COOH groups, NH₂ groups and the like,

R₂=hydrogen, alkyl or aryl groups; the alkyl or aryl groups may includeother groups such as COOH groups, NH₂ groups and the like;

wherein

R₁=hydrogen, alkyl or alkoxy groups, wherein at least one RI is analkoxy group; the alkyl or alkoxy groups may include other groups suchas COOH groups and the like,

R₂=hydrogen, alkyl or aryl groups; the alkyl or aryl groups may includeother groups such as COOH groups, NH₂ groups and the like; and

wherein

R₁=alkyl, alkoxy or aryl groups, the alkyl, alkoxy and aryl groups mayinclude COOH groups and the like,

R₂=alkyl or aryl groups, the alkyl and aryl groups may include COOHgroups, NH₂ groups and the like.

Administration of the imaging material to a human or other organism viamethods including ingestion, injection (either alone or with a carriersuch as an emulsifier, liposomes, or other biocompatible materials),inhalation or direct topical or internal application is followed bymovement of the imaging material through the tissues of the organism,and concentration of the imaging material in pathological tissue,relative to surrounding tissue. The pathology or target tissue may beoptically imaged, delineated, detected or diagnosed by: (i) illuminatingthe area containing the preferentially localized imaging material withlight at any wavelength absorbed by the imaging material; and (ii) theabsorption or the emission of light through fluorescence orphosphorescence or the generation of heat by this material. Detection ofabsorption or emission of light can be by the human eye,photomultiplier, photosensitive diode, or by any suitable device fordetection of light at the appropriate wavelengths. Heat can be detectedby photothermal, photoacoustic, mirage effect, or other calorimetricmethods. Alternatively, if the agent is a magnetic resonance contrastagent, detection is by magnetic resonance imaging instrumentation. Ifthe agent contains a radioactive isotope, radiation from this isotopecan be detected. If the agent is an X-ray contrast material, pathologyor target tissue can be imaged and identified using X-ray radiologicaltechniques.

The optical agent, after excitation with light, will form excitedsinglet states, and may form excited triplet states or other high-energystates. In general, such states can react chemically with the organism,causing tissue damage, or react with oxygen to form harmful singletoxygen or radical species, or with other molecules that may be presentto produce other reactive, high energy species than may in turn reactwith the organism, causing tissue damage. In the imaging material, theauxiliary chromophore quenches the excited state or other high-energystate of the agent via an energy or electron transfer process. Thisquenching process produces a low-energy state, such as the polyenetriplet state, or singlet state, which is incapable of harming theorganism, and is incapable of sensitizing the formation of singletoxygen or other reactive species. The method of coupling the auxiliarychromophore to the agent and the proper choice of agent and auxiliarychromophore ensure that this quenching is a rapid process. The method ofcoupling the auxiliary chromophore to the agent is facilitated throughthe overlap of electronic orbitals which may occur by either chemicalbonding, Van der Waals interaction, or by using a coupling compound suchas a protein to hold the auxiliary chromophore and the agent in nearproximity. This rapid quenching deactivates the high energy triplet orother state of the agent before it can interact deleteriously withtissue, sensitize singlet oxygen formation, or react with othermolecules that may be present to form a harmful species. In essence, theauxiliary chromophore allows the agent to perform as an imaging ordetection agent, but prevents it from significantly damaging theorganism.

In this invention, a method of administering an imaging material thatwill preferentially localize in pathology or target tissue and absorband emit light without damaging the mammalian host upon irradiation withlight comprises the steps of administering a diagnostically effectiveamount of the imaging materials and allowing said imaging material tocirculate and accumulate and localize in pathology or target tissue,preferably from about 3 to 48 hours prior to the diagnostic or surgicalprocedure, and exposing the mammalian host to light causing the imagingmaterial to absorb and fluoresce and thereby permitting visualizationand definition of the pathology or target tissue to be removed ortreated, or using some other visualization method such as thosedescribed hereinabove.

More specifically, the imaging material employed in the practice of thisinvention localizes in pathology or target tissue, absorbs light of onewavelength, may emit light of another wavelength, but does not damagehealthy tissue. The process of this invention may be used both fordiagnosis and as a valuable adjunct to surgery as light emitted from thepathology or target tissue by the localized agent in the imagingmaterial would sharply define the location of the pathology or targettissue to be removed.

In practice, an imaging material is administered intravenously to amammalian host in a dosage of from 0.5 to 50 μmol/kg of body weight from1 to 72 hours prior to exposure to radiation having a wavelength of fromabout 600 to about 1,100 nanometers. The imaging materials may beconveniently administered either solubilized in a biocompatible emulsionsuch as a Tween-80, CREMOPHOR EL emulsion (Sigma Chemical Company) orother suitable lipophilic emulsion or incorporated into liposomes suchas unilamellar or multilamellar liposomes of a synthetic lipid such asdipalmitoylphosphatidylcholine (DPPC) sold by Sigma Chemical Company,Inc. After the photosensitive imaging material has had sufficient timeto circulate and localize in pathology or target tissue, the mammalianhost is exposed to a light so that the imaging material localized inpathology or target tissue absorbs and fluoresces permittingvisualization of the pathology or target tissue location, size andconfiguration. The most suitable electromagnetic radiation sources, e.g.light sources, are those that emit radiation at wavelengths of between600 to about 1,100 nanometers. The imaging agents of this invention havetheir red-most absorption bands in the range of about 600 to 1,100nanometers. The use of imaging materials that absorb electromagneticradiation in the 600-1,100 nm region permits the diagnostic light topenetrate the tissue more deeply and image deeper pathology or targettissue tissues than can be obtained with molecules that absorb only atshorter wavelengths. Thus, monochromatic radiation at these wavelengthswould be preferentially absorbed. In addition to using the human eye asa detector, a light-sensitive electronic device such as aphotomultiplier or photodiode array could be used as a detector toprovide a picture or electronic image of localized material.Alternatively, any other detection and visualization method for theagent may be employed. Even if light is not part of the diagnosticprocess, the auxiliary chromophore will protect the skin and othertissues of the organism from photodamage by adventitious light absorbedby the agent.

EXAMPLES

To further assist in the understanding of the present invention and notby way of is limitation, the following examples are presented. In theexamples reported below, the ¹H NMR spectra were obtained at 300 to 500MHZ and used ≦1% solutions in chlorolorm-d with tetramethylsilane as aninternal reference. The UV-vis spectra were recorded on a HewlettPackard 8450A spectrophotometer. For transient absorption studies,samples were placed in 1 cm×1 cm×4 cm cuvettes and deoxygenated bybubbling with argon. The apparatus used for the transient absorptionwork features excitation with ca. 5 ns pulses of less than approximately5 mJ at 590-700 nm. An adequate signal-to-noise ratio was achieved bysignal averaging (typically about 500 flashes). The details of thespectrometer are described by Gust et al. J. Am. Chem. Soc. 1986, 108,8028, incorporated by reference herein. Fluorescence decay measurementswere made on ca. 1×10⁻⁵M solutions using the time-correlated singlephoton counting method. The excitation source was a frequency-doubled,mode-locked Nd-YAG laser coupled to a synchronously pumped, cavitydumped dye laser with excitation at 590 arm. Detection was via amicrochannel plate photomultiplier (Hamamatsu R2809U-01), and theinstrument response time was ca. 35 ps.

Example 1

(Carotenophthalocyanines with short carotenoids)

Carotenophthalocyanines consisting of a nine-double bond carotenoidpolyene (the auxiliary chromophore) covalently linked to a syntheticzinc-phthalocyanine (the agent)

wherein

R₁=hydrogen, substituted or unsubstituted alkyl or alkoxy group,

R₂=hydrogen, substitued or unsubstitued alkyl or aryl group, asexemplified by the chemical compound

have been synthesized as follows:

A portion of 4-nitrophthalonitrile (5.15 g, 29.8 mmole) was dissolved in125 mL methanol in a hydrogenation flask. The catalyst, 10% Pd/C (515mg), was added to the mixture which was thin flushed with H₂ and stirredunder 50 psi of H₂ for 1 hour. The catalyst was removed by filtrationand the solvent was distilled off under vacuum. The desired4-aminophthalonitrile was obtained in 86% yield (3.668 g).

4-Aminophthalonitrile (3.60 g, 25.2 2.mmole) was dissolved in 8.6 mL ofpyridine. Hexanoyl chloride (8.6 10, 61.5 mmole) was added and themixture was stirred under N₂ for 2 hours. The pyridine was distilledunder vacuum until a yellow-orange residue was obtained. This residuewas dissolved in dichloromethane and washed with 0.1 M HCl (5×15 mL) andthen neutralized with aqueous sodium bicarbonate (3×50 mL). The organicsolution was separated and dried over magnesium sulfate. Purification bycolumn chromatography (silica, dichloromethane: methanol 5%) afforded4.3 g (71% yield) of 4-hexanamidophthalonitrile as a light yellowcompound.

Catechol (13.21 g, 120.1 mmole) was dissolved in 120 mL ofdimethylformamide (DMF). Potassium carbonate (41.42 g) and butyl bromide(49.22 g) were added to the solution. The mixture was stirred under N₂for 24 hours at 95° C. Excess potassium carbonate and butyl bromide (30g of each) were added and the mixture was stirred overnight. Anotheraddition of 30 g of butyl bromide and further stirring overnight wasnecessary to complete the reaction. The mixture was diluted with I L ofether and washed with water (6×250 mL) and then dried over magnesiumsulfate. The ether was removed by distillation under vacuum, yielding aviscous brownish liquid. The liquid product was purified by distillationunder vacuum. The fractions with a boiling point between 69-73° C. werecollected. A yellow liquid identified as 1,2-dibutoxybenzene (19.05 g,71.5% yield) was obtained.

A portion of 1,2-dibutoxybenzene (19.0 g, 85.6 mmole) was dissolved in175 mL of dichloromethane. The solution was cooled to 0° C. and keptunder N₂ while Br₂ (9.6 mL 186.8 mmole) dissolved in 25 mLdichloromethane was added slowly. Half of the Br, solution was addedwhile the temperature was kept at 0° C. and the other half was addedafter the temperature was raised to 25° C. The reaction mixture wasstirred for 2 hours under N₂. The work-up consisted of a wash withaqueous sodium hydrogen sulfate until the yellow color disappeared,followed by two washes with sodium bicarbonate solution. The organicsolution was dried over sodium sulfate and filtered. Evaporation of thesolvent afforded 31.71 g of 1,2-dibromo-4,5-dibutoxybenzene as a clearliquid (97.5% yield).

A portion of 1,2-dibromo-4,5-dibutoxybenzene (31.7 g, 83.5 mmole) wasdissolved in DMF, 22.8 g (254.6 mmole) of cuprous cyanide was added andthe suspension was stirred and heated to reflux under N₂ for 6 hours.The mixture was cooled to room temperature and poured into 1.1 L ofammonium hydroxide (30%), whereupon a blue liquid with a green frothformed. Air was bubbled through the liquid for 20 hours. The solid wasremoved by filtration, washed with 10% ammonium hydroxide until thefiltrate was clear, and then washed with water to neutral pH. The greenpowder was extracted with hexanes in a Soxhlet extractor for one week.The solvent volume was reduced to 200 mL, whereupon a solid formed. Thepowder was collected and recrystallized giving 10.53 g (46% yield) ofwhite crystals of 4,5-dibutoxyphthalonitrile.

4-Hexanamidophthalonitrile (1.467 g, 6.1 mmole) and4,5-dibutoxyphthalonitrile (2.48 g, 9.1 mole) were dissolved in 80 mL of2-dimethylaminoethanol and stirred under an ammonia flow at 100° C. for2 hours 1.8-diazabicyclo[5.40] undecene-7 (DBU) (1.3 mL) was added andthe stirring was continued for 15 minutes. Zinc acetate (1.146 g) wasadded and the mixture was stirred at 100° C. overnight. The greenmixture was cooled to room temperature and 60 mL water: methanol (1:1)was added. The precipitate formed was collected, washed with water anddried. The product was purified by column chromatography (silica,chloroform: methanol 1%). Zinc9,10,16,17,23,24-hexabutoxy-2-hexanamidophthalocyanine, was obtained in18.5% yield (790 mg). ¹H-NMR (500 MHZ, DMSO-CDCl₃) δ1.02 (3H, t,—CO(CH₂)₄ CH ₃), 1.12-1.20 (18H, m, —OCHCH₂CH₂ CH ₃), 1.47-1.53 (4H, m,—CO(CH₂)₂ CH ₂ CH ₂CH₃), 1.70-1.80 (12H, m,—OCH₂CH₂ CH ₂CH₃), 1.87 (2H,q, —COCH₂ CH ₂(CH₂)₂CH₃), 2.00-2.10 (12H, m, —OCH₂ CH ₂CH₂CH₃), 2.63(2H, t, —COCH ₂(CH₂)₃CH₃), 4.47-4.55 (12H, m, —O—CH ₂CH₂CH₂CH₃), 8.40(1H, d, J=8 Hz, Pc 3-H), 8.45-8.60 (6H, m, Pc 8,11,15,18,22,25-H), 9.10(1H, d,J=8 Hz, Pc 4-H), 9.60 (1H, d, Pc 1-H), 10.6(1H, s, —NHCO—); MSm/z: 1437.3 (M+2)⁺; UV/vis (95% CH₂Cl₂— 5% CH₃OH) 352, 454, 616, and 683nm.

The phthalocyanine obtained in the reaction above (290 mg, 0.26 mmole)was dissolved in 40 mL of tetrahydrofuran (THF) and 40 mL of a saturatedmethanolic solution of KOH. The solution was heated to 75° C. andstirred overnight under N₂. An extra amount of THF (10 mL) was added andthe mixture was stirred overnight. The reaction mixture was diluted withchloroform, the solution was washed with water (3×), the organic layerwas dried over sodium sulfate and filtered, and the solvent wasdistilled. The desired zinc2-amino-9,10,16,17,23,24-hexabutoxyphthalocyanine (214 mg) was obtainedin 81% yield.

A portion of ethyl-β-apo-8′-carotenoate (62 mg, 0.13 mmole) prepared inaccordance with O. Isler et al., Helv. Chim. Acta, 1959, 42, 864, wasdissolved in 14 mL of THF. A solution of 1.2 g of KOH in 10 mL ofmethanol was added and the mixture was stirred under N₂ overnight. THF(2 mL) was added and the stirring was continued overnight. The reactionmixture was diluted with chloroform and neutralized with 20% acetic acidand washed with water. The solvent was distilled under vacuum.β-apo-8′-carotenoic acid (49 mg) was obtained in 84% yield.

The carotenoic acid prepared above (49 mg, 0.11 mmole) was dissolved in6 mL of toluene and 2 mL of pyridine. One drop of thionyl chloride wasadded and the mixture stirred under N₂ for 15 minutes. The solvent wasdistilled under vacuum and the residue was kept under vacuum for anextra 15 minutes. The residue was redissolved in 2.5 mL of pyridine and6 mL of dry chloroform. The aminophthalocyanine prepared above (100 mg,0.098 mmole) dissolved in 6 mL chloroform was added to the solution ofthe acid chloride and the mixture was stirred overnight under N₂. Thereaction mixture was diluted with chloroform, washed with water anddried under vacuum. The product was purified by column chromatography(silica, chloroform-methanol 0.2%). A second column (silica, chloroform)was necessary to obtained 73 mg (52% yield) of pure material shown inFIG. 1. ¹H-NMR (500 MHZ, DMSO-CDCl₂) δ1.01-1.04 (6H, m, Car 16-CH₃, Car17-CH₃), 1.16-1.21 (18H, m, —OCH₂CH₂CH₂ CH ₃), 1.46 (2H, m, Car 2-CH₂),1.59 (2H, m, Car 3-CH₂), 1.71 (3H, s, Car 18-CH₃),1.75-1.83 (12H,m,—OCH₂ CH ₂CH₃),1.96 (3H, s, Car 19-CH₃), 2.00 (3H, s, Car 20-CH₃),2.08 (15H, m,—OCH₂ CH ₂CH₂CH₃, Car 20′-CH₃), 2.32 (3H, s, Car 19′-CH₃),4.50-4.57 (12H, m, —O—CH₂CH₂CH₂CH₃), 6.16-6.8.1 (11H, m, Car ═CH—), 7.44(1H, d, J=10 Hz, Car 10′-H), 8.45-8.6 (7H, m, Pc 4,8,11,15,18,22,25-H),9.1 (1H, d,J=8 Hz, Pc 3-H), 9.63 (1H, s, Pc 1-H), 10.4 (1H, S, —NHCO—);MS m/z: 1437.3 (M+2)⁺; UV/vis (95% CH₂Cl₂— 5% CH₃OH) 352, 454, 616, and683 nm.

These carotenophthalocyanines are designed to be administered byinjection or other means, to localize preferentially in pathology ortarget tissue tissue, and to absorb light, especially in the 700 nmregion, and thereupon fluoresce at a longer wavelength than theabsorbance wavelength in accordance with the general description above.The use of diagnostic light having a wavelength in the 700 nm regionallows for deeper penetration of the light, for imaging deeper pathologyor target tissue tissues, and for reducing tissue damage relative tolight at shorter wavelengths. Light absorption, fluorescence, or bothcan be detected by either eye or instrumental methods, thus identifyingand demarcating the pathology or target tissue tissue.

This class of carotenophthalocyanines bears relatively short, 9 doublebonds or fewer, carotenoid moieties, which serve to enhance thefluorescence yield relative to related molecules with longer carotenoidpolyenes. Furthermore, these compounds are designed to quench thephthalocyanine triplet states by triplet-triplet energy transfer to theattached carotenoid. This will prevent formation of singlet oxygen byenergy transfer from the porphyrin triplet states, and thus preventdamage to healthy tissue caused by singlet oxygen, or long-livedporphyrin triplet states. These molecules are also designed to bereadily excreted by normal tissue, including the liver and spleen.

Example 2

(Type B carotenopurpurins)

Molecules consisting of one or more carotenoid polyenes (the auxiliarychromophore) covalently linked to a type B purpurin (the agent) such as

wherein

R₁=hydrogen, substituted or unsubstituted alkyl or alkoxy groups; eachR₁ may be the same or different,

R₂=hydrogen, substituted or unsubstituted alkyl or aryl groups, each R₂may be the same or different,

as exemplified by

have been synthesized as follows:

The starting porphyrin:5,15-bis-(4-carbomethoxyphenyl)-2,8,12,8-tetraethyl-3,7,13,17-tetramethylporphyrinwas prepared according to A. Osuka T. Nagata, F. Kobayashi and K.Maruyarna, Heterocyclic Chem., 1990, 21, 1657-1659. The pure compoundwas obtained in 17% yield. ¹H-NMR (300 MHZ, CDCl₃) δ-2.21 (2H, brs,—NH), 1.77 (12H, t, —CH₂ CH ₃) 2.47 (12H, s, —CH₃), 4.02 (8 H, q, —CH₂CH₃), 4.14 (6H, S, —CO₂ CH ₃), 8.12 (4H, d, Ar—H), 8.45 (4H, d, Ar—H),10.26 (2H, s, H meso); MS m/z: 747.3 (M+H)⁺ (Calc. M=746.4); UV/vis(CH₂Cl₂) 235, 408, 508, 542, 576, 628 nm.

To a 1 L round bottomed flask equipped with a stirring bar was added 1.0g (1.34 mmole) of the porphyrin prepared above and 400 mL of toluene. Tothe suspension was added 3.0 g (11 mmole) of nickel acetoacetate(Ni(AcAc)₂.H₂O) and the temperature was raised to 100° C. for 48 hours.After this period all the free base porphyrin was converted to thenickel analog. The solvent was partially removed and the pasty solid wasredissolved in chloroform. The solution was washed with water, driedover anhydrous sodium sulfate and filtered, and the solvent wasevaporated. The residue was recrystallized from CHCl₃/CH₃OH to afford1.05 g (97%) of pure nickel5,15-bis-(4-carbomethoxyphenyl)-2,8,12,8-tetraethyl-3,7,13,17-tetramethylporphyrin.¹H-NMR (300 MHZ, CDCl₃) δ 1.60 (12H, t, —CH₂ CH ₃), 2.21 (12H, s, —CH₃),3.68 (8 H, q, —CH ₂CH₃), 4.08 (6H, S, —CO₂ CH ₃), 7.96 (4H, d, Ar—H),8.32 (4H, d, Ar—H), 9.44 (2H, s, H meso); MS m/z: 803.5 (M+H)⁺ (Calc.M=802.3); UV/vis (CH₂Cl₂) 235, 351, 406, 530, 564 nm.

A Vilsmeier reagent was prepared as follows. Twenty mL of DMF was addedto a 250 mL round bottomed flask equipped with a stirring bar and athree-way adapter. The flask and its contents were cooled to 5-10° C.before and during the dropwise addition of 10 mL of POCl₃. The resultingviscous liquid was stirred at room temperature under an atmosphere of N₂for an additional 25 minutes.

At the same time that the Vilsmeier reagent was being prepared, 1.0 g(1.37 mmole) of the nickel porphyrin was dissolved in 600 mL of1,2-dichloroethane and cooled to 5-0° C. The Vilsmeier reagent was addeddropwise to the porphyrin solution over a 10 minute period. The stirringwas continued for 10 more minutes at 5° C. and then at room temperaturefor 2.5 hours. During this period the solution turned from a red colorto green. Aqueous sodium bicarbonate with an excess of solid sodiumbicarbonate was added to the reaction mixture with caution. Thetwo-phase mixture was warmed to 40° C. and kept at this temperature for16 hours with vigorous stirring. The green organic solution wascollected and concentrated under vacuum. The residue was redissolved indiethyl ether and washed with water, dried over anhydrous sodium sulfateand then concentrated. The product was chromatographed on silica gel(CH₂Cl₂: acetone 0.5-2%) to yield 1.08 g (94% yield) of the desirednickel meso-formylporphyrin. ¹H-NMR (300 MHZ, CDCl₃) δ 1.40-1.60 (12H,m, —CH₂ CH ₃), 1.99 (6H, s, —CH₃), 2.02 (6H, s, —CH₃), 3.40-3.60 (8H, m,—CH ₂CH₃), 4.06 (6H, s, —CO₂ CH ₃), 7.82 (4H, d, Ar—H), 8.29 (4H, d,Ar—H), 9.03 (1H, s, H meso), 11.23 (1H, s,—CHO; MS m/z: 831.3 (M+H)⁺(Calc. M=830.3); UV/vis (CH₂CL₂) 330, 438, 668 nm.

To a 250 mL round bottomed flask equipped with a condenser and a threeway adapter was added 3.76 g (10.0 mmole) ofN,N-diethylformamidomethyltriphenylphosphonium bromide and 100 mL ofo-xylene. The mixture was stirred under N₂ as 0.4 g (10.0 mmole) of NaH(60% oil dispersion) was added. The stirring was continued until all thephosphonium salt reacted. At this point the nickel meso-formylporphyrinprepared above (0.416 g, 0.50 mmole) was added and the reaction mixturewas warmed to reflux for 30 hours. After cooling the reaction mixture,the solvent was removed under vacuum and the residue was redissolved indichloromethane. The solution was washed with citric acid and then withaqueous sodium bicarbonate. The solvent was evaporated and the residuewas chromatographed on silica gel (CH₂Cl₂: acetone 2-4%) to give 427 mg(92% yield) of the desired nickel meso-acrylamidoporphyrin. ¹H-NMR (300MHZ, CDCl₃) δ 0.86 (3H, t, —NCH₂ CH ₃), 1.17 (3H, t, —NCH₂ CH ₃),1.45-1.58 (12H, m, —CH₂ CH ₃), 2.05 (6H, s, —CH₃), 2.11 (6H, s, —CH₃),3.10 (2H, q, —NCH ₂CH₃), 3.43-3.61 (10 H, m, —CH ₂CH₃ and —NCH ₂CH₃),4.06 (6H, s, —CO₂ CH ₃), 5.56 (1H. s, vinyl-H), 7.89 (4H, br, Ar—H),8.30 (4H, d, Ar—H), 9.17 (1H, s, H meso), 9.77 (1H, d, vinyl-H); MS m/z:928.5 (M+H)⁺ (calc. M=927.4); UV/vis (CH₂Cl₂) 316, 426, 550, 586 nm.

To a 250 mL round bottomed flask equipped with a stirring bar and athree way adapter was added 0.4 g (0.43 mmole) of the nickelmeso-acrylamidoporphyrin prepared as above and 70 mL of trifluoroaceticacid. The dark solution was stirred at room temperature under a N₂atmosphere for 1 hour. The reaction mixture was diluted withdichloromethane and washed with water until neutral. The solvent wasevaporated and the residue was chromatographed on silica gel (CHCl₃:methanol 3%) to give 342 mg (91%) of the desired free basemeso-acrylamidoporphyrin. ¹H-NMR (300 MHZ, CDCl₃) δ-2.00 (2H, s, —NH),1.11 (3H, t, —NCH₂ CH ₃), 1.20-1.40 (9H, m, —CH₂ CH ₃ and —NCH₂ CH ₃),1.57 (6H, t, —CH₂ CH ₃), 2.06 (6H, s, —CH₃), 2.23 (6H, s, —CH₃),3.30-3.80 (12H, m, —CH ₂CH₃ and —NCH ₂CH3), 4.11 (6H, s, —CO₂ CH ₃),6.41 (1H, d, vinyl-H), 8.19 (4H, d, Ar—H), 8.43 (4H, d, Ar—H), 9.63 (1H,s, H meso), 10.24 (1H, d, vinyl-H); MS m/z: 872.1 (M+H)⁺ (Calc.M=871.5); UV/vis (CH₂Cl₂) 236, 340, 438, 530, 602 nm.

The purpurin was prepared from the free base meso-acrylamidoporphyrinfollowing an accordance with Gunter et al., Australian J. Chem., 1990,4, 1839-1860. To a 100 mL round bottomed flask equipped with a stirringbar and a three way adapter was added 0.2 g (0.23 mmole) of the freebase meso-acrylamidoporphyrin, 39 mL o-dichlorobenzene and 2.6 mLpiperidine. The solution was flushed with N₂ and the reaction mixturewas warmed to 120° C. under a N₂ atmosphere. After 24 hours, thereaction mixture was cooled and diluted with dichloromethane. Thissolution was washed with diluted HCl and then with sodium bicarbonate.The organic layer was concentrated to a dark solid and this material waschromatographed on silica gel (CH₂Cl₂: acetone 5-8%) to give 90 mg (45%yield) of the expected purpurin diester. ¹H-NMR (300 MHZ, CDCl₃) δ −0.46(1H, s, —NH), −0.26 (3H, t, —CH₃), 0.50 (1H, s, —NH), 1.26-1.73 (16H, m,Pur-CH₃, —NCH₂ CH ₃ and Pur 23-CH ₂CH₃) 2.12 (3H, s, Pur-CH₃), 2.23 (3H,s, Pur-CH₃), 2.25 (3H, s, Pur-CH₃), 2.53 (Pur 23-CH ₂CH₃), 3.30-3.90(10H, m, —CH ₂CH₃ and —NCH ₂CH₃), 4.07 (3H, s, —CO₂ CH ₃), 4.10 (3H, s,—CO₂ CH ₃) 4.53 (1H, q, Pur 18-H), 8.44-7.37 (8H, m, Ar—H), 8.61 (1H, s,Pur 21-H), 9.58 (1H, s, Pur 11-H meso); MS m/z: 872.3 (M+H)⁺ (Calc. M871.5); UV/vis (CH₂Cl₂) 237, 287, 314, 432, 538, 576, 634, 690 nm.

To a 250 mL round-bottomed flask equipped with a stirring bar and athree way adapter was added 0.1 g (0.115 mmole) of purpurin diester, 100mL of THF and 20 mL of methanol. The solution was stirred under anitrogen atmosphere as 3 mL of 10% aqueous KOH was added. After theaddition of the base the solution was warmed to 40° C. for 24 hours.Thin layer chromatography (TLC) indicated that a single polar materialhad formed. The reaction mixture was poured into chloroform and washedwith diluted citric acid. The organic layer was further washed withwater (×2) and concentrated, and the residue was dried under vacuum. Thepurpurin diacid was used without further purification in the followingstep.

To a 100 mL round-bottomed flask equipped with a stirring bar and athree way adapter was added 80 mg (0.095 mmole) of the purpurin diacid,25 mL of benzene and 153 μL (1.90 mmole) of pyridine. The mixture wasstirred under nitrogen as 69 μL (0.948 mmole) of thionyl chloride wasadded dropwise. The stirring was continued at room temperature for 1hour. After that time the solvent was removed under reduced pressure. Analiquot of benzene was added and also distilled under reduced pressure.The residue was redissolved in dichloromethane (20 mL) that contained150 μL of pyridine. To this mixture was added7′-apo-7′-(4-aminophenyl)-β-carotene (96 mg, 0.190 mmole), as describedin Gust et al., Methods in Enzymology, 1992, 213, 87-100. After stirringfor 15 minutes, methanol (1 mL) was added to the reaction mixture andthe stirring was continued for an additional 15 minutes. The contents ofthe flask were diluted with dichloromethane (100 mL) and the solutionwas washed with dilute citric acid and then with aqueous sodiumbicarbonate. Once dried over anhydrous sodium sulfate, the solvent wasremoved and the residue was chromatographed on silica gel withtoluene/6-10% ethyl acetate as the solvent. Further purification bycolumn chromatography on silica gel with dichloromethane/1-3% acetoneafforded 8 mg (5% yield) of the desired dicarotenopurpurin. ¹H-NMR (500MHz, CDCl₃) δ −0.18 (1 H, brs, —NH), 0.28 (3H, t, 24-CH₃), 0.55 (1H,brs, —NH), 1.04 (12 H, s, Car 16-CH₃, Car 17-CH₃), 1.34 (6H, t, —NCH₂ CH₃), 1.40-1.70 (17H, m, Car 2-CH₂, Car 3-CH₂, Pur 3-CH₂ CH ₃. Pur 9-CH₂CH ₃, Pur 1 3-CH₂ CH ₃), 1.72 (6H, s, Car 18-CH₃), 1.98-2.01 (18H, 3s,Car 19-CH₃, Car 20-CH₃, Car 20′-CH₃), 2.08 (6H, s, Car 19′-CH₃), 2.10(3H, s, Pur-CH₃), 2.20 (1H, m, Pur 19-CH₂CH₃), 2.22 (3H, s, Pur-CH₃),2.36 (3H, S, Pur-CH₃), 2.65 (1H, m, Pur 19-H₂CH₃), 3.40-400 (10H, m, Pur3-CH ₂CH₃, Pur 9-CH ₂CH₃, Pur 13-CH ₂CH₃, —NCH ₂CH₃), 4.66 (1H, s, Pur25 ═CH—), 5.50 (1H, s, Pur 25 ═CH—), 6.10-7.00 (28H, m, Car ═CH—),7.50-8.40 (16H, m, Pur Ar—H, Car Ar—H), 8.51. (1H, s, Pur 21-H), 9.59(1H, Pur 11-H meso); MS m/z: 1819.4 (M+2)⁺; UV/vis (CH₂Cl₂) 446, 478,512, 584, 642, 702 nm.

These type B carotenopurpurins are designed to be administered byinjection or by other means to localize preferentially in pathology ortarget tissue tissue, to absorb light at 700 arm or greater, especiallyat 710 nm, and thereupon fluoresce at a longer wavelength than theabsorbance wavelength in accordance with the general description above.The use of diagnostic light having a wavelength in the 700 nm regionallows for deeper penetration of the light, for imaging deeper pathologyor target tissue tissues, and reduced tissue damage relative to agentsabsorbing only at shorter wavelength. Light absorption, fluorescence orboth can be detected by either eye or instrumental methods, thusidentifying and demarcating the pathology or target tissue tissue.Furthermore, these compounds are designed to quench the purpurin tripletstates by triplet-triplet energy transfer to the attached carotenoid.This will prevent formation of singlet oxygen by energy transfer fromthe purpurin triplet states, and thus prevent damage to healthy tissuecaused by singlet oxygen, or long-lived purpurin triplet states.

Example 3

Carotenoporphyrins consisting of carotenoid polyene, the auxiliarychromophore, covalently linked to a synthetic porphyrin (the agent)bearing one or several methoxygroups

wherein

R₁=hydrogen or substituted or unsubstituted alkyl or alkoxy group; eachR₁ may be the same or different, wherein at least one RI is an alkoxygroup;

R₂=hydrogen, substituted or unsubstituted alkyl or aryl group.

as exemplified by

have been prepared as follows:

The required porphyrin,5-(4-acetamidophenyl)-10,15,20-tris(3,5-dimethoxyphenyl)porphyrin, wasprepared in accordance with Gust et al., Methods in Enzymology, 1992,213, 87-100. UV/vis (CH₂Cl₂) 421, 516, 550, 591, 645 nm.

A portion of this porphyrin (1.2 g, 1.41 mmole) was dissolved in amixture of 150 mL of THF, 150 mL of methanol and 90 mL of aqueous KOH(40%). The mixture was stirred at 65° C. for 24 hours under anatmosphere of N₂. The crude reaction was diluted with 200 mL ofdichloromethane and washed with water until neutral. Chromatography onsilica gel with dichloromethane and increasing amounts of acetone (0.1%to 1.5%) afforded 794 mg (70% yield) of5-(4-aminophenyl)-10,15,20-tris(3,5dimethoxyphenyl)porphyrin. ¹H-NMR(300MHz, CDCl₃) δ −2.79 (2H, s,—NH), 3.96 (18H, s, —OCH₃), 6.90 (3H, t,J=2.3 Hz, 10,15,20Ar 4-H), 7.05 (2H, d, J=8.3 Hz 5Ar 3,5-H), 7.40 (6H,d, J=2.3 Hz, 10,15,20Ar 2,6-H), 7.98(2H, d, J=8.3 Hz, 5Ar 2,6-H), 8.92(8H, s, meso); MS m/z: 810.2 (M+H)⁺ (Calc. M=809.3).

The carotenoid acid chloride was prepared from7′-apo-7′-(4-carboxyphenyl)-β-carotene in accordance with Gust et al.,Methods in Enzymology, 1992, 213, 87-100. A portion of7′-apo-7′-(4-carboxyphenyl)-β-carotene (590 mg, 1.11 mmole) wasdissolved in 130 mL of dry toluene and 30 mL of dry pyridine. Thissolution was stirred under an atmosphere of N₂ while 240 μL of thionylchloride was added. The mixture was kept well stirred for 30 minutes atroom temperature. The solvent and excess thionyl chloride were distilledunder vacuum and the residue was kept under high vacuum forapproximately 30 minutes. During this time, 384 mg (0.475 mmole) of theaminoporphyrin prepared above was dissolved in 130 mL of dry toluene and30 mL of dry pyridine. The carotenoid acid chloride was dissolved in 10mL of toluene and 1 mL of pyridine. This solution was added to theporphyrin solution and the mixture was stirred under an atmosphere of N₂for 24 hours at room temperature. The crude reaction mixture was dilutedwith dichloromethane and extracted with a saturated solution of sodiumbicarbonate. Column chromatography on silica gel with dichloromethaneand increasing amounts of ethyl acetate (0.1% to 2%) gave 490 mg of purematerial as shown in FIG. 3 (78% yield). ¹H-NMR (300 MHz, CDCl₃) 5-2.80(2H, s,—NH), 1.04 (6H, s, Car 16-CH₃,Car 17-CH₃), 1.44-1.52 (2H, m, Car2-CH₂), 1.57-1.68 (2H, m, Car 3-CH,), 1.72 (3H, s, Car 18-CH₃), 1.99(3H, s, Car 19CH₃), 2.00 (3H, s, Car 20-CH₃), 2.01 (3H, s, Car 20′-CH₃),2.02-2.04 (2H, m, Car 4-CH₂), 2.09 (3H, s, Car 19′-CH₃), 3.96 (18H, s,—OCH₃), 6.10-6.80 (13H, m, Car ═CH—), 6.90 (3 H, t, J=2.1 Hz, 10,15,20Ar4-H), 7.06 (1H, d, J=16 Hz, Car 8′-H), 7.41 (6H, d, J=2.1 Hz, 10,15,20Ar2,6H), 7.62 (2H, d, J=8 Hz, Car 1′,5′-H), 7.98 (2H, J=8 Hz, Car2′,4′-H), 8.04 (2H, d, J=8.4 Hz, 5Ar 3,5-H), 8.16 (1H, s, —NH), 8.21(2H, d, J=8.4 Hz, 5Ar 2,6-H), 8.91 (8H, m, meso); MS m/z: 1325.4 (M)⁺(Calc. M=1325.7); UV/vis (CH₂Cl₂) 422, 481, 513, 589, 646 nm.

These polymethoxycarotenoporphyrins are designed to be administered byinjection or other means, to localize preferentially in pathology ortarget tissue tissue, to absorb light, especially in the 650 nm region,and thereupon fluoresce at a longer wavelength than the absorbancewavelength in accordance with the general description above. Lightabsorption, fluorescence or both can be detected by either eye orinstrumental methods, thus identifying and demarcating the pathology ortarget tissue tissue. Furthermore, these compounds are designed toquench the porphyrin triplet states by triplet-triplet energy transferto the attached carotenoid. This will prevent formation of singletoxygen by energy transfer from the porphyrin triplet states, and thusprevent damage to healthy tissue caused by singlet oxygen, or long-livedporphyrin triplet states. These molecules are also designed to bereadily excreted by normal tissue, including the liver and spleen.

Example 4

(Octa-alkyl carotenoporphyrins)

Carotenoporphyrins consisting of a carotenoid polyene (the auxiliarychromophore) covalently linked to a synthetic octa-alkyl porphyrin (theagent)

wherein

R₁=hydrogen, substituted or unsubstituted alkyl, alkoxy or aryl group;each R₁ may be the same or different,

R₂=hydrogen, substituted or unsubstituted alkyl or aryl group;

as exemplified by

have been synthesized as follows:

To a 50-mL flask under a nitrogen atmosphere were added 46 mg (0.086mmole) of 7′-apo-7′-(4-carboxyphenyl)-β-carotene, 12 mL ofdichloromethane, 10 μL (0.091 mmole) of N-methylmorpholine, and 16 mg(0.091 mmole) of 2-chloro-4,6-dimethoxy 1,3,5-triazine. The mixture wasstirred for 2 hours, and 50 mg (0.057 mmole) of the required aminoporphyrin prepared in accordance with Kuciauskas et al., J. Phys. Chem.B., 1997, 101, pp. 429-440, was added, along with 10 μL (0.091 mmole) ofN-methylmorpholine and 1 1 mg (0.091 mmole) of4-N,N-dimethylaminopyridine. After stirring for 17 hours, aqueous sodiumbicarbonate was added, and the mixture stirred for 1.5 hours.Dichloromethane was added, and the resulting mixture was washed withwater, dilute aqueous citric acid, and dilute aqueous sodiumbicarbonate. Sodium sulfate was used to dry the organic phase, and afterfiltering, the solvent was distilled at reduced pressure. Chromatographyof the residue on silica gel (dichloromethane containing 1-2% acetone)gave crude product, which was recrystallized from dichloromethane andmethanol to give 57 mg of the compound shown in FIG. 4 (71% yield).¹H-NMR (300 MHz, CDCl₃) δ −2.40 (2H, brs, —NH), 1.04 (6H, s, Car 16-CH₃,Car 17-CH 3), 1.43-1.52 (2H, m, Car 2-CH₂), 1.57-1.68 (2H, m, Car3-CH₂), 1.73 (3H, s, Car 18-CH₃), 1.78 (12H, m, 2-CH₃, 8-CH₃, 12-CH₃,18-CH₃), 1.99 (3H, s, Car 19-CH₃), 2.00 (3H, s, Car 20-CH₃), 2.01 (3H,s, Car 20′-CH₃), 2.01 (2H, m, Car 4-CH₃), 2.10 (3H, s, Car 19′-CH₃),2.56 (6H, s, 13-CH₃, 17-CH₃), 2.59 (6H, s, 3-CH₃, 7-CH₃), 4.02 (3H, s,ArOCH₃), 4.02 (8H, m, 2-CH₂, 8-CH₂, 12-CH₂, 18-CH₂), 4.07, (3H, s,ArOCH₃), 6.1-6.8 (13H, m, Car ═CH—), 6.84 (2H, AB, J=9 Hz, Naphthyl2,3-H), 7.07 (1H, d, J=16 Hz, Car 8′-H), 7.62 (2H, d, J=8 Hz, Car1′,5′-H), 7.97-8.23 (12H, m. Car 2′,4′-H, 5Ar2,3,5,6-H, 15Ar2,3.5,6-H,Naphthyl 7-H, NH), 8.42 (1H, d7 J=9 Hz, Naphthyl 8H.), 8.44 (1H, brs,—NH), 8.90 (1H, brs, Naphthyl 5-H), 10.24 (2H, s, 10-CH, 20-CH); MS m/z1393 (M+H)⁻; UV/vis (CH₂Cl₂) 410, 450 (sh), 484, 512, 574, 626 nm.

These carotenoporphyrins are designed to be administered by injection orother means, to localize preferentially in pathology or target tissuetissue, and to absorb light, especially in the 630 nm region, andthereupon fluoresce at a longer wavelength than the absorbancewavelength in accordance with the general description above. Lightabsorption, fluorescence or both can be detected by either eye orinstrumental methods, thus identifying and demarcating the pathology ortarget tissue tissue.

This class of carotenoporphyrins also contains an important structuraldifference from other carotenoporphyrins. For steric reasons, thepresence of the β-pyrrolic alkyl groups flanking the meso-aromatic ringsbearing the carotenoid moiety limits the π—π overlap between thefluorophore and the carotenoid. This structural change serves to isolatethe chromophores, resulting in increased fluorescence. Thus, thisimaging material presents a novel method for enhancing the detection ofpathology or target tissue tissue.

Furthermore, these compounds are designed to quench the porphyrintriplet states by triplet-triplet energy transfer to the attachedcarotenoid. This will prevent formation of singlet oxygen by energytransfer from the porphyrin triplet states, and thus prevent damage tohealthy tissue caused by singlet oxygen, or long-lived porphyrin tripletstates. These molecules are also designed to be readily excreted bynormal tissue, including the liver and spleen.

Example 5

4-Nitroquinoline-1-oxide (4-NQO) induces dyplastic lesions and squamouscell carcinoma of the rat palate. The stage of dysplasia correlates withthe 4-NQO application period. The compound synthesized in Example 3 wasinjected in rats in which the palate was treated with 4-NQO for 0, 6, 12or 18 weeks. The compound was administered in a liposome preparation oran emulsion at a dosage of 5.3 micromole compound/kg.

Fluorescence images and emission spectra were taken before and atseveral timer intervals after injection. After injection, thefluorescence in the palate tumor tissue increased homogeneously anddecreased very slowly. Two peaks were observed which correspond to thein vitro fluorescence emission peaks of the compound synthesized inExample 3. Thus, it is clear that the compound was incorporated into thetumor tissue and was responsible for the observed fluorescence. Themaximum in fluorescence measured 10 hours post injection was the samefor the animals treated with 4-NQO for 0, 6 and 12 weeks. The rats thatwere treated with the tumor-inducing agent for 18 weeks, when the tumorwas well-developed, showed a statistically significant higherfluorescence maximum than the rats treated for 0, 6 or 12 weeks.

Example 6

Ultraviolet B (UV-B) light induces skin cancer in hairless mice.Hairless mice were exposed to UV-B light and developed visible skintumors. The compound synthesized in Example 3 was administered to UV-Bexposed mice as well as unexposed mice in a liposome preparation or asan emulsion at a dosage of 5.3 micromol/kg. Fluorescence images andemission spectra were taken before and at several times after injection.Two peaks were observed in the fluorescence spectra after injectionwhich correspond to the in vitro fluorescence emission peaks of thecompound synthesized in Example 3. After injection of the compound, thefluorescence in healthy skin increased, and the fluorescence in thetumor tissue increased much more, indicating that the compound wastransported to the tissues of the tumor and preferentially localizedthere. After three days, localization of fluorescence in tumors wasstill observable. The maximum fluorescence in normal, i.e., non UV-Btreated skin, occurred between 10 hours and 20 hours after injection. InUV-B treated skin with no visible macroscopic tumors, maximumfluorescence occurred between 10 and 20 hours after injection. Invisible tumor tissue, the maximum fluorescence also occurred between 10and 20 hours post injection and was at least four times more intensethan the fluorescence from skin without tumors. Thus, using this method,pathology or target tissues may be readily delineated from thesurrounding skin by visual observation, photography or other imagingmethods.

While not wishing to be bound by any one theory, it is thought thatphotoprotection by these agents arises when the carotenoid auxiliaryagent rapidly quenches thecyclic tetrapyrrole triplet state by atriplet-triplet energy transfer mechanism. This returns the cyclictetrapyrrole to the normal unexcited ground electronic state andproduces the carotenoid triplet state. The carotenoid triplet state istoo low in energy to produce singlet oxygen, and returns harmlessly tothe carotenoid ground state with liberation of heat.

Although the invention has been described herein with reference tospecific embodiments, many modifications and variations therein willreadily occur to those skilled in the art. Accordingly, all suchvariations and modifications are included within the intended scope ofthe invention.

The invention claimed is:
 1. A compound having the structure: